The sinoatrial node (SA node or SAN) is a finely-tuned structure that initiates and sets the rhythm of the heartbeat. Recent insights into embryonic development have pinpointed T-box (Tbx) transcription factors as key determinants of SA node development. Tbx18, in particular, has been shown to be indispensable for the specification of the SA node during development. However, little is known about Tbx-driven gene regulatory pathways which specify morphogenesis of the SA node, and how these pathways lead to automaticity in pacemaker cells. We seek to test the general hypothesis that re-expression of Tbx18 suffices to reprogram postnatal cardiomyocytes to pacemaker cells. We propose to reveal Tbx18-dictated gene regulatory pathways that give rise to de novo automaticity. In parallel, we will characterize the changes in electrophysiological pathways which confer automaticity on normally-quiescent ventricular myocytes, and compare the reprogrammed mechanisms of pacing to those which are operative in native SA nodal myocytes, as the gold standard for genuine pacemaker cells. The main impediment to understanding the gene regulatory pathways to automaticity is a lack of a system to study specific targets of SA nodal transcriptional regulatory pathways. This is because the rapid temporal and spatial changes during embryonic development make it difficult to study specific targets of transcriptional regulation. In contrast, our proposed studies in postnatal cardiomyocytes offer a milieu for relatively slow-changing (neonatal) or steady-state (adult) electrophysiology. AIMs 2 and 3 are designed to gain insights into the Tbx18-reprogrammed automaticity in single-cell, two-cell pacing unit, 2D monolayers, and 3D structures. Our cell culture systems could readily be applied for other transcription factor- or disease-mediated studies of cellular electrophysiology. Three scientific innovations are imminent from this study. One, data from AIMs 1 and 2 will provide insights into molecular determinants of automaticity as quiescent myocytes begin to beat spontaneously and autonomously upon Tbx18 re-expression. Two, outcomes of AIM 3 will provide important insights into the source-sink mismatch phenomenon in SAN physiology. Three, at the conclusion of the proposed studies, a candidate for a biological pacemaker could be identified as an alternative to electronic pacemaker devices. Furthermore, Genome wide association studies (GWAS) have identified and linked T-box transcription factor genes with congenital heart defects and conduction system abnormalities. Dysregulation of Tbx18-guided pathways may cause improper morphogenesis of conduction system and may lead to arrhythmias. Knowledge gained from AIMs 1, 2, and 3 will provide the first cause-effect explanations for clinical manifestations of these arrhythmias. PUBLIC HEALTH RELEVANCE: Abnormally slow or fast heart rhythms, known as cardiac arrhythmias, affect many in North America and the number of people affected by this disease is increasing steadily with our aging populace. A key to treating these pathologic conditions is a fundamental understanding of the cardiac rhythm generation. We propose a detailed mechanistic study to investigate the initiation and propagation of a heartbeat, which will lead to better understanding and treatment of cardiac arrhythmias.